Sequencing and analysis of the complete mitochondrial genome of Ochotona coreana from China and its phylogenetic analysis

Abstract The complete mitogenome sequence of Ochotona coreana was determined using long PCR. The genome was 17,283 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, 1 origin of L strand replication, and 1 control region. The overall base composition of the heavy strand is A (31.5%), C (28.7%), T (26.2%), and G (13.6%). The base compositions present clearly the A–T skew, which is most obviously in the control region and protein-coding genes. Mitochondrial genome analyses based on MP, ML, NJ and Bayesian analyses yielded identical phylogenetic trees. This study verifies the evolutionary status of O. coreana in Ochotonidae at the molecular level. The mitochondrial genome would be a significant supplement for the O. coreana genetic background. The seven Ochotona species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

Ochotona coreana was promoted to a species from a subspecies O. hyperborea coreana . In this paper, the complete mitochondrial genome of O. coreana was sequenced for the first time on ABI 3730XL using a primer walking strategy and the long and accurate PCR, with five pairs of long PCR primers and with 14 pairs of sub-PCR primers. A muscle sample was obtained from a female O. coreana captured from the Dahailin regions of Changbaishan Mountains in Heilongjiang Province, China (44 19 0 59 00 N, 128 12 0 10 00 E). The muscle tissue was preserved in 95% ethanol and stored at À75 C before use. The specimen and its DNA is stored in Animal and Plant Herbarium of Mudanjiang Normal University. The voucher number is CBST2019001.
The control region of O. coreana mitochondrial genome was located between the tRNA-Pro and tRNA-Phe genes, and contains only promoters and regulatory sequences for replication and transcription, but no structural genes. Three domains were defined in O. coreana mitochondrial genome control region (Zhang et al., 2009): the extended terminationassociated sequence (ETAS) domain, the central conserved domain (CD) and the conserved sequence block (CSB) domain.
The total length of the protein-coding gene sequences was 11,398 bp. Most protein-coding genes initiate with ATG except for ND2, ND3, and ND5, which began with ATC or ATT. Seven protein-coding genes terminated with TAA. The incomplete stop codons (T--) were used in COX3, ATP6, ND3 and ND4. A strong bias against A at the third codon position was observed in the protein-coding genes. The frequencies of CTA (Leu), ATT (Ile), TTA (Leu), and ATA (Met) were higher than those of other codons. The length of tRNA genes varied from 59 to 75 bp. Twenty-one of them could be folded into the typical cloverleaf secondary structure except the tRNA-Ser (AGY), whose complete dihydrouridine arm was lacking.
Most O. coreana mitochondrial genes were encoded on the H strand, except for the ND6 gene and eight tRNA genes, which were encoded on the L strand. Some reading frame intervals and overlaps were found. One of the most typical was between ATP8 and ATP6. The L-strand replication origin (OL) was located within the WANCY region containing five tRNA genes (tRNATrp, tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr). This region was 33 bp long and had the potential to fold into a stable stem-loop secondary structure. The total base composition of O. coreana mitochondrial genome was A (31.5%), C (28.7%), T (26.2%), and G (13.6%). The base compositions clearly present the A-T skew, which was most obviously in the control region and protein-coding genes.
In order to explore the evolution of Lagomorpha species which include Ochotonidae and Leporidae, especially the evolution of genus Ochotona from China, here, we investigate the molecular phylogenetics of Chinese O. coreana using the complete mitochondrial genome sequence of 21 species. All sequences generated in this study have been deposited in the GenBank (Figure 1).
Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees, indicating a close phylogenetic affinity of species. The phylogram obtained from the maximum parsimony method is shown in Figure 1. It shows that two major phyletic lineages were present in Lagomorpha: Ochotonidae and Leporidae.

Disclosure statement
No potential conflict of interest was reported by the author(s).